Sensitive non-radioactive nucleic acid hybridization assay for plum pox virus detection

© INSTITUTPASTEuR/ELsEVIER Paris 1994

Res. Virol.

1994, 145, 387-392

Sensitive non-radioactive nucleic acid hybridization assay for plum pox virus detection L. Palkovics, J. Burgy~tn and E. Bal,~izs Agricultural Biotechnology Center, H 2101 GSdOl15 Szent, GySrgyi A. u. 4 (Hungary)

SUMMARY

A new non-radioactive sandwich hybridization assay was designed to simplify the analysis of a large number of plant samples. Plant material was homogenized in 0.5% SDS and added directly to the hybridization reaction, in which a pair of identifying probes were used. One of the probes was biotinylated capture RNA specific for plum pox virus (PPV) strain SK-68; the other RNA probe was synthesized from a plasmid bearing the adjacent sequence of this strain and was labelled with digoxigenin (DIG). Both purified viral RNA and crude extracts from PPV-infected plants were used as target for sandwich hybridization. The hybridization reaction was carried out in a streptavidin-coated EUSA plate. After extensive washing, the viral RNA was detected by conventional colour reaction using anti-DIG/alkaline phosphatase conjugate. In comparative experiments, we have shown that this non-radioactive detection system is more sensitive than conventional ELISA techniques and we were able to detect virusspecific RNA in more than 50 % of the EUSA-negative samples.

Key-words: ELISA, RNA-RNA hybridization, Plum pox virus; Biotin, Digcrxigenin, Streptavidin, Sensitivity, Potyvirus.

INTRODUCTION Different ELISA techniques are currently used throughout the world as routine diagnostic tools for plant virus identification. Several companies have antisera against all types of plant pathogens. Plant quarantine laboratories and plant breeders are being equipped with ELISA readers and with all the necessary hardware for the utilization of this advanced technique. However, this widespread ELISA technique is not applicable for the detection of a number of plant pathogens. V'troids, for example, have no coat proteins. Some plant

Submitted April 19, 1994, accepted May 17, 1994

viruses, like tobacco rattle virus, are not good immunogens. Several important viruses, besides inducing severe diseases, are present in low titres in plant tissue, and some plant protein might interact with the antigen-antibody reactions. To overcome these difficulties, several types of nucleic acid hybridization are being used in plant virus detection (Bar-Joseph et al., 1985, 1986; Maule et al., 1983; Baulcombe et al., 1984; Hull and AI-Hakim, 1988; Habili et al., 1987; Rouhainen et al., 1991). A three-component hybridization test has been developed for nucleic acid detection and identification from crude samples (Syv~nen,

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1986; Syv~inen et al., 1986; Ranki et al., 1983). This test requires two adjacent, but not overlapping DNA reagents, subcloned into different vectors. One is immobilized on a solid support and acts as a catching probe, while the other is labelled to enable the detection. When the target nucleic acid (either DNA or RNA) is present in the test, it anneals to both reagents and forms a sandwich hybrid. If a poly-A÷ RNA is the catching reagent, oligo-dT or a poly-U paper can be used. The plum pox virus is present at very low concentrations in infected trees and degrades very quickly in crude woody plant extracts. Therefore, in this paper, we provide details on an RNARNA sandwich hybridization assay for the detection o f this virus.

MATERIALS AND METHODS Virus and viral RNA purification The plum pox virus (PPV) SK-68 strain was propagated in Nicotiana clevelandii L. and grown under normal greenhouse conditions for 14-21 days after inoculation. Virus was purified from these plants essentially as described by Lain et al. (1988). RNA isolation and fractionation were carried out as described by Brakke and Van Pelt (1970). cDNA synthesis and molecular cloning The first cDNA strand was primed with oligo-dT and the dscDNA (double-stranded eDNA) was synthesized using the "Amersham eDNA Synthesis Kit" according to the manufacturer's instructions. The eDNA was ligated into S m a I linearized, dephosphorylated pUC18 or pUC19 plasmids, and then used to transform Escherichia coli DH5ot competent cells (Maniatis et al., 1982). The pPPV-72 clone containing 2,400 bp from the 3' end of the viral sequence (Palkovics et al., 1993) was selected for the development of the detection assay. Subcloning was carried out with H i n d l I I , S a c I and B a m H I restriction endonucleases (Amersham).

BIO = biotin. cDNA -- complementaryDNA. DIG = digoxigenin.

Sample preparation Plant extracts were prepared by grinding 0.5 g leaves in 5 ml 0.5 % SDS in individual plastic bags containing gauze. Crude plant extracts were collected in Eppendorf tubes.

Preparation of RNA probes The RNA probes used for this study were biotinylated, digoxigenin-labelled or radiolabelled.

Biotin (BIO) labelling of the RNA The 200-ml transcription reaction mix contained 2 ~tg HindlII linearized plasmid, 20 ~tl 10 x tran-

scription buffer, 100 V human placental ribonuclease inhibitor (Amersham), 0.5 mM ribonucleotide mix ahd 60U T7 RNA polymerase (Amersham). The reaction mix was incubated at 37°C for 1 h. The DNA template was eliminated with 5U RQ I DNase (Promega) at 37°C during a 20-rain incubation. After stopping the reaction, the RNA was purified on a "Bio-Gel P-30" (Bio-Rad) column and with phenol-chloroform and precipitated with ethanol. The precipitated RNA was resuspended in sterile water at 1 ~g/p.1 final concentration. The same volume of photobiotin-acetate (Bresa) was added in the dark on ice and irradiated from a distance of 10 cm with a 1000-W mercury lamp (HgMI 1000/D1 Tungsram) for 15 rain; 0.1M Tris-HC1 pH 9.0 was added to the reaction mix to increase the volume to 100 ~tl. The photobiotinylated RNA was extracted by butanol essentially as described by Foster et al. (1985). The labelled RNA wasprecipitated and dissolved in sterile water. Digoxigenin (DIG) labelling of the RNA The reaction mixture (100 ml) contained 1 gg SacI linearized plasmid DNA, 10 lxl 10× transcrip-

tion buffer, 60 U human placental ribonuclease inhibitor (Amersham), 10 ~tl 10xDIG-dbonucleotide mix (Boerhinger) and 40 U T7 RNA polymerase (Amersham). The mixtures were incubated at 37°C for 20 min. The DNA template was eliminated by adding 5U RQ I DNAse (Promega) and incubating at 37°C for 20 min; 100~tl of 0.5% SDS solution

dscDNA = double-strandedcDNA. PPV -- plumpox virus. SDS = sodiumdodecylsulphate.

SENSITIVE N O N - R A D I O A C T I V E N U C L E I C ACID H Y B R I D I Z A T I O N A S S A Y

were added to the reaction mixture and purified on a Bio-Gel P-30 (Bio-Rad) column and with phenolchloroform and precipitated with ethanol. Radiolabeiling of the R N A

The reaction mix (50 ~tl) contained 1 ~tg SacI linearized plasmid DNA, 5~tl 10x transcription buffer, 40 U human placental r i b o n u c l e a s e inhibitor (Amersham), 0.5 mM ribonucleotide mix without CTP, 50 I.tM CTP, 0.8 MBq ot-32p-CTP and 40U T7RNA polymerase (Amersham). The reaction mix was incubated at 37°C for 1 h. RNA was purified on Bio-Gel P-30 (Bio-Rad) column. Hybridization on nylon m e m b r a n e s

The crude plant extracts were denaturated by adding an equal volume of denaturation buffer (50 % formamide, 20 mM Hepes, 1 mM EDTA pH 8.0, 6 % formaldehyde). Ten p.1 were spotted onto a nylon membrane (Hybond-N, Amersham). Hybridization conditions were according to Maniatis et aL (1982). ELISA The ELISA assays were carded out according to the double antibody sandwich method using "Bioreba PPV" antisera. Hybridization on E L I S A plate

ELISA plates (Nunc Maxi Sorp) were sensitized overnight at 4°C with 150 I.tl (10 I.tl/ml) streptavidin (Immunoselect) solution in 50 mM Tris pH 7.5. Immediately before using the sensitized plates, they were extensively washed four times for 1 min with a PBS solution containing 0.05 % Tween-20 and twice PBS. In each reservoir a 40-~tl sample was incubated with 80 ~tl hybridization mix (40mM Na phosphate buffer pH 7.0 4×SSC, lxDenhardt's solution, 20% deionized formamide, 2% dextran sulphate, 0.5% SDS, 10 ~tg yeast RNA and 101° biotinylated RNA probe or 101° DIG-labelled RNA probe). Samples were covered with 60 Ixl paraffin oil and incubated at 60°C for 4 h and additionally for 2 h at 37°C in a rotary shaker (Dynatech). After incubation, the wells were washed with 200 ~tl 0.5% SSC and 0.2% SDS at 60°C, and three times for 2 min with PBS containing 0.05 % Tween-20 at 37°C. After this extensive washing, 120 p.l anti-DIG/alkaline phosphatase conjugate was added to each reservoir in a dilution of 1:1000 and incubated at 37°C for 4 h. After incubation, the same washing procedure was applied as described above (3x2 min PBS + 0.05 % Tween-20).

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For colorimetric detection, 120 ~tl of substrate solution (p-nitrophenylphosphate, 1 tablet in 30 ml substrate buffer; Sigma) was added to each well and incubated overnight at room temperature in the dark. Extinction values were measured using a "Labsystem Multiscan Plus" ELISA reader at 405 ran.

RESULTS pPPV-72 of PPV c D N A was subcloned into two transcription vectors (fig. 1). The pAM18/DIG plasmid contains the 670-bp part of the pPPV-72 clone in the pAM18 transcription plasmid. The p A M 1 8 / B I O plasmid contains the longer part (1760bp) of the pPPV-72 clone. From these two clones, we synthesized BIO-labelled and DIGlabelled RNA. R N A transcripts complementary to the viral R N A were synthesized by T7 R N A p o l y m e r a s e , after linearization of the p A M 1 8 / D I G p l a s m i d b y S a c I , and the pAM19/BIO plasmid by HindlIl. T h e R N A transcripts were intact, of correct size and approximately 10-20 ~tg R N A transcripts were synthesized from 1 ~tg template (fig. 1). Purified viral R N A from PPV strain SK-68 was used to d e t e r m i n e the sensitivity o f this method. Ten-fold dilutions of the purified viral R N A were prepared in healthy N. clevelandii L. crude plant extracts and used in this experiment. Each virus dilution was tested four times. The same healthy crude plant extract served as a negative control, and PPV-infected N. clevelandii L. sap was used as a positive control. The sandwich hybridization method is able to detect 107 viral R N A molecules with good accuracy (OD 0.147) as shown in table I. This corresponds to ca 50 pg of viral RNA, while the sensitivity of molecular hybridization using 32p-labelled c R N A probes is 0.2 to 1 pg viral R N A depending on the P P V strain (Varveri et al., 1987). Healthy crude plant sap has very little or no effect on the sensitivity of this assay (table I). To test the superiority o f this double-sandwich nucleic acid hybridization assay over the conventional ELISA method, a series of plant samples were analysed using different techniques on the same samples. Leaves collected from naturally i n f e c t e d fruit trees w e r e cut into f o u r e q u a l

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Table I. Sensitivity of the sandwich hybridization method. Viral RNA molecules (*) 101° 109

108 107 Healthy N. clevelandii Infected N. clevelandii

OD 1.257 0.828 0.239 0.188 0.019 0.438

1.289 0.728 0.228 0.167 0.001 0.372

Mean value 1.300 0.910 0.171 0.140 0.021 0.308

1.253 0.700 0.222 0.093 0.003 0.453

1.275 0.792 0.215 0.147 0.011 0.393

(*) Purified viral RNA from PPV strain SK-68 (Palkovics et al., 1993) was used to determine the sensitivity of this method. Ten-fold dilutions of the purified viral RNA were prepared in healthy N; clevelandii L. crude plant extract and used in this experiment.

pieces : 2 opposite parts were used for sap extraction for ELISA and 2 for hybridization assays. Forty ELISA-negative samples were analysed

with the sandwich hybridization technique and with radiolabelled nucleic acid hybridization assays (table I1). The same plant extract was used

SENSITIVE N O N - R A D I O A C T I V E N U C L E I C A C I D H Y B R I D I Z A T I O N A S S A Y

Table H. Comparison of the sandwich hybridization and radioactive hybridization methods for PPV detection: analysis of ELISA-negative samples from naturally infected trees.

Plant species Myrobalan Wild peach Almond Apricot Total

Number of samples 18 11 10 1 40

Number of positive samples: sandwich- radioactive hybridihybridization zation 13 1 5 1 20 (50%)

16 8 10 1 35 (87.5 %)

for both hybridization assays. More then 50% of the ELISA-negative samples proved to be positive for PPV when tested by the sandwich hybridization test. With radiolabelling of the probe RNA, the number of positive samples was even higher, i.e. 87.5 % of the ELISA-negative samples.

DISCUSSION

In human medical diagnosis, several detection kits have recently been used that are based on nucleic acid h y b r i d i z a t i o n . In c o n v e n t i o n a l nucleic acid hybridization assays, the immob'dized nucleic acids were detected on a filter by a radioactive probe. This is a simple assay with a slow

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reaction rate. Solution hybridization methods are more efficient, faster and easier (Syvanen et aL, 1986). Our diagnostic assay utilizes the advantages of solution hybridizations. We have used RNA probes, because the RNA-RNA hybrids are more stable than the DNA-RNA hybrids. The first step in this method is the formation of the sandwich hybrids that include the viral RNA, the BIO-labelled capture RNA and the DIG-labelled probe RNA. The hybridization is carried out at a high temperature (60°C) and under denaturation condition to ensure efficient and specific hybrid formation (fig. 2). In the second step, the hybrids formed are collected on a streptavidin-coated ELISA plate at a lower temperature (37°C). The next step is intensive washing of the ELISA plate several times at a higher temperature. After this step, the anti-DIG/alkaline phosphatase conjugate is b o u n d to the DIGlabelled RNA, followed by a colour reaction. This method is much more sensitive than conventional ELISA, although it does not achieve the sensitivity of radioactive hybridization. Because the radiolabelled probes can be used for only a short time due to the short half-life of the isotope, and considering the further risk of hazardous waste generated working with the isotope, we e s t a b l i s h e d this alternative n o n - r a d i o a c t i v e hybridization system. The assay described here for PPV detection did not require specific sample treatment; it combined different steps of recently published detection systems for identification of this virus (Var-

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veri et al., 1988; Wetzel et al., 1990, Korschineck et al., 1991). Its m a j o r a d v a n t a g e c o m p a r e d to published and currently used detection systems is its easy adaptation to large-scale screening o f the virus. This m e t h o d can be introduced in any routine E L I S A laboratory, and can be extended as a model for other virus identification as well.

Acknowledgements This work was supported by grants from OMFB (9197-07-0113) and Phare Accord (0384). We thank Dr. M~u-iaK61ber for performing the ELISA test.

M~thode sensible, non radioactive, de d~tection du virus de la sharka par hybridation mol~culaire U n e m 6 t h o d e de d 6 t e c t i o n non r a d i o a c t i v e , d6sign6e sous le nom de technique d'hybridation "ARN-ARN en sandwich" a 6t6 raise au point pour analyser des &:hantillons de plantes infect6es par le virus de la sharka ou PPV. Le mat6del viros6, broy6, est ajout6 au milieu d'hybridation renfermant 2 ribosondes adjacentes marqu6es, l'une par la biotine, l'autre par la digoxig6nine. Le m61ange d'hybridation r e n f e r m a n t la cible (c'est-~-dire de I ' A R N viral purifi6 ou bien le jus brut pr6par6 h partir de plantes infect6es) est ensuite d6pos6 dans les puits d ' u n e plaque ELISA pr6alablement incub~e en pr6sence de streptavidine. Apr~s incubation, I'ARN viral, pris en sandwich entre les deux ribosondes est r6v616 par des anticorps anti-digoxig6nine marqu6s par la phosphatase alcaline. Des tests comparatifs rnontrent que la sensibilit6 de cette m6thode de d6tection est nettement sup6fieure h celle des tests ELISA classiques: 50 % des 6chantillons n6gatifs en ELISA sont positifs lorsqu'ils sont 6tudi6s par cette nouvelle m&hode. Mots-clds: ELISA, Hybridation ARN-ARN, Virus de la sharka; Biotine, Digoxig6nine, Streptavidine, Sensibilit6, Potyvirus.

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Bar-Joseph, M., Segev, D., Buckle, W., Yesodi, V., Franck, A. & Rosner, A. (1986), Application of synthetic DNA probes for the detection of viroids and viruses, in "Developments and applications in virus testing/association of applied biologists" (R.C.C. Jones & L. Torrance) (pp. 13-23). The Lavenham Press Ltd, Lavenham. Baulcombe, D.C., Boulton, R.W., Flavell, R.B. & Jellis, GJ. (1984), Recombinant DNA probes for detection of viruses in plants, "British Crop Protection Conference - Pests and Disease" (pp. 207-213). Brakke, M.K. & Van Pelt, N. (1970), Linear-log gradient for estimating sedimentation coefficients of plant viruses and nucleic acids. AnaL Biochem., 38, 56-64. Forster, A.C., Mclnnes, J.L., Skingle, D.C. & Symons, R.H. (1985), Nonradioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin. Nucl. Acis Res., 13, 745-761. Habili, N., McInnes, J.L. & Symons, R.H. (1987), Nonradioactive, photobiotin-labelled DNA probes for the routine diagnosis of barley yellow dwarf virus. J. Virol. Methods, 16, 225-237. Hull, R. & AI-Hakim, A. (1988), Nucleic acid hybridization in plant virus diagnosis and characterization. Trends BiotechnoL, 6, 213-218. Koschineck, I., Himmler, G., Sagl, R., Steinkeller, H. & Katinger, W.D. (1991), A PCR membrane spot assay for the detection of plum pox virus in bark of infected trees. J. Virol. Methods, 31, 139-146. Lain, S., Riechmann, J.L., M6ndez, E. & Garcia, J.A. (1988), Nucleotide sequence of the 3'terminal region of plum pox potyvirus RNA. Virus Res. 10, 325-342. Maniatis, T., Fritch, E.F. & Sambrook, J. (1982), Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York. Made, AJ., Hull, R. & Donson, J. (1983), The application of spot hybridiTation to the detection of DNA and RNA viruses in plant tissue. J. Virol. Methods, 6, 215-224. Palkovics, L., Burgy~in,J. & Ball~zs, E. (1993), Comparative sequence analysis of four complete primary structures of plum pox virus. Virus Genes, 7, 339-347. Ranki, M., Palva, A., Virtanen, M., Laaksonen, M. & S0derlund, H. (198~), Sandwich hybridization as a convenient method for detection of nucleic acids in crude samples. Gene, 21, 77-85. Rouhainen, L., Laaksonen, R., Karjalainen, R. & S6dedund, H. (1991), Rapid detection of a plant virus by solution hybridization using oligonucleotide probes. J. Virol. Methods, 34, 81-90. Syv~inen, A.-C., Laaksonen, M. & SOderiund, H. (1986), Fast quantification of nucleic acid hybrids by affinity-based collection. Nucl. Acid Res., 14, 5037-5048. Syvfinen, A.-C. (1986), Nucleic acid hybridization: from research tool to routine diagnostic method. Med. Biol. 64, 313-324. Varveri, C., Candresse, T., Cugnsi, M., Ravelonandro, M. & Dunez, J. (1988), Use of 32p-Iabelled transcribed RNA for dot hybridization detection of plum pox virus. Phytopathology, 78, 1280-1283. Wetzel, T., Tavert, G., Teycheney, P.Y., Ravelonandro, M., Candresse, T. & Dunez, J. (1990), Dot hybridization detection of plum pox virus using 32P-labeled RNA probes representing nonstructural viral protein genes. J. ViroL Methods, 30, 161-172.